Wee1 knockdown by siRNA treatment is specific and its effects are kinase dependent. NCI-H1299 vector control cells were treated with no siRNA (a), non-targeting siRNA (N1) (b), Wee1 #6 (c) or Wee1 #8 (d) siRNAs for five days. The remaining percentage of viable cells after proliferation is given for each sample. (e) NCI-H1299 vector control cells were subjected to 5 nM siRNA treatment for two days following plating and analyzed by Western blot to determine effects on Wee1 knockdown and pCDK levels. The ratio of phospho/total CDK (p/t CDK) resulting from each siRNA treatment was determined by densitometry relative to their Actin controls. (f-i) NCI-H1299 cells over-expressing a wild type Wee1 with siRNA resistant seed regions. The samples are the same as in a-e. (k-o) NCI-H1299 cells over-expressing a kinase-altered Wee1 K328R with siRNA resistant seed regions. The samples are the same as in a-e. All results are representative of three separate experiments.